RNAseq for cultured Control and TgAPPsweOCN BMSCs (sorted Ai9+ Osteoblast-linage cells)
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PROVIDER: PRJNA776261 | ENA |
REPOSITORIES: ENA
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Project description:Purpose: To investigate if and how APPswe in OCN-Cre+ OB-lineage cells gives rise to the brain and behavior phenotypes, we purified OCN-Cre+ BMSCs (marked by tdTomato+, believed to be OB progenitors) from both the 6-MO control (OCN-Cre; Ai9) and TgAPPsweOCN; Ai9 mice using fluorescence-activated cell sorting (FACS) and then subjected them to RNA-seq analysis Methods: (1)the whole bone marrow cells of 6-month-old OCn-Cre;Ai9 andTgAPPswe;OCn-Cre;Ai9 flushed out from long bones of mice with DMEM were filtered through a 70-mm filter mesh, washed, re-suspended, and then plated in 100-mm dishes with growth medium (DMEM plus 10% FBS), which were incubated at 37oC with 5% CO2. The non-adherent cells were removed 72 hours after changing the medium. The attached bone marrow cells were cultured with the growth medium for 7 days. These cells were then resuspended and plated at culture dishes and cultured for another 3-6 days with the same growth medium. These cells, so called BMSCs. (2)Then cell media were removed from culture dishes and cells were rinsed with PBS. Trypsin solution was added to incubate at 37°C for 2 min. The detached adherent cells were centrifuged, and the pellet cells were washed with 1 ml cold PBS, and finally resuspended in 0.5 ml PBS with 1% FBS for flow cytometry analysis. Flow cytometric analysis was performed by use of a flow cytometer in CWRU core facility. Acquisition and analysis were performed by using FACSDiva 8.0.1 software (BD).(3)Total RNAs were extracted from purified Td+ OB-progenitor cells from OCN-Cre; Ai9 and TgAPPsweOCN; Ai9 mice by flow cytometer. RNA Integrity Number (RIN) was accessed for every sample, and the samples were considered qualified with RIN> 2. These RNA samples were then subjected to RNA-seq analyses by BGI America (Cambridge, MA) using the DNBseq platform. Firstly, we removed the reads mapped to rRNA and obtained the raw data with 52.47 Mb reads. After filtering low-quality, adaptor-polluted and high content of unknown base reads in the sequencing reads, 51.9 Mb clean reads were obtained per sample on average. Then clean reads were mapped to reference genome using HISAT2. On average 92.91% reads were mapped and the uniformity of the mapping result for each sample suggests that the samples were comparable. Comparisons to RNAseq were normalized to fpkm Results: 917 up- and 1825 down-regulated genes were identified in APPswe+ OB progenitors. Among these genes, 154 up- and 269 down-regulated genes encode secreted proteins. Interestingly, go analysis showed that most up-regulated genes are involved in inflammatory response, cytokine production, and cytokine/chemokine-mediated signaling pathways; and most down-regulated genes are implicated in cell cycle, cell proliferation, and bone mineralization. Conclusions: Increased cytokines and chemokines in APPswe+ OB-progenitors, indicating that APPswe+ OB progenitors may undergo cellular senescence.
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